Physiological and Biochemical Characterization of a Novel Nicotine-Degrading Bacterium Pseudomonas geniculata N1

Management of solid wastes with high nicotine content, such as those accumulated during tobacco manufacturing, poses a major challenge, which can be addressed by using bacteria such as Pseudomonas and Arthrobacter. In this study, a new species of Pseudomonas geniculata, namely strain N1, which is capable of efficiently degrading nicotine, was isolated and identified. The optimal growth conditions for strain N1 are a temperature of 30°C, and a pH 6.5, at a rotation rate of 120 rpm min−1 with 1 g l−1 nicotine as the sole source of carbon and nitrogen. Myosmine, cotinine, 6-hydroxynicotine, 6-hydroxy-N-methylmyosmine, and 6-hydroxy-pseudooxynicotine were detected as the five intermediates through gas chromatography-mass and liquid chromatography-mass analyses. The identified metabolites were different from those generated by Pseudomonas putida strains. The analysis also highlighted the bacterial metabolic diversity in relation to nicotine degradation by different Pseudomonas strains.


Introduction
Nicotine, a principal pyridine alkaline in tobacco plants, is notorious for its significant contribution to tobacco addiction. However, nicotine is very toxic to humans because it is easily absorbed in the body; its hydrophilic nature contributes to the environmental contamination [1]. Moreover, large quantities of tobacco wastes containing high concentration of nicotine are produced during tobacco manufacturing process. These wastes have been classified as ''toxic and hazardous wastes'' by European Union Regulations [2]. In addition, the American Medical Association has issued a public strategy strengthening the forcible reduction of nicotine level in tobacco [3]. As an environmentfriendly treatment, microbial degradation of nicotine has been considered as a promising method due to its low cost and high efficiency.
In this study, a novel strain, Pseudomonas geniculata N1, capable of degrading nicotine was isolated. Along with the identification and characterization of this new nicotine-degrading strain, we also determined the optimal conditions for cell growth and nicotine degradation. Compared with other Pseudomonas and Arthrobacter species, strain N1 exhibited a distinct color change, during its growth with nicotine as the sole source of carbon and nitrogen. The intermediates of strain N1-mediated nicotine degradation were identified by high-performance liquid chromotography (HPLC), ultraviolet (UV) absorption, gas chromatography mass (GC-MS), and liquid chromatography mass (LC-MS) analysis. The data showed that strain N1 decomposes nicotine via a unique pathway, which is different from those reported by Pseudomonas strains. This study suggests that the nicotine-degrading bacterium has future potential application on the treatment of the waste generated during tobacco manufacturing. The findings might help further the research for characterizing the molecular mechanisms underlying nicotine degradation by strain N1.

Chemicals and media
L-(-)-Nicotine ($99% purity) was purchased from Fluka Chemie GmbH (Buchs, Switzerland). All other chemicals were of analytical grade. The ''nic medium'' was a minimal medium containing 13.3 g K 2 HPO 4 ?3H 2 O, 4 g KH 2 PO 4 , 0.2 g MgSO 4 ?7H 2 O and 0.5 ml of trace elements solution. L-(-)-Nicotine was added to this minimal medium after filtration sterilization to a final concentration of 1 g l 21

Strain identification and characterization
After the extraction of genomic DNA by the Wizard Genomic DNA purification kit (Promega Corp., Madison, WI, USA), 16S rRNA gene was amplified by PCR with the universal primer pair of 27F (59-AGAGTTTGATCCTGGCTCA-39) and 1492R (59-GGTTACCTTGTTACGACTT-39). PCR amplification was carried out with pfu polymerase (Tiangen, Beijing, China) by denaturation at 94uC for 5 min, followed by 30 cycles of 94uC for 30 s, 60uC for 30 s, and 72uC for 3 min. The PCR product was purified for sequence analysis and homology alignment analysis using the BLAST search program (http://www.ncbi.nlm.nih.gov/ BLAST.html). A phylogenetic tree was constructed with the neighbor-joining (NJ) method using MEGA 4.1 [19].
A series of experiments were conducted for simultaneously identifying the morphological, physiological and biochemical characteristics of the strain. The morphology was studied using a transmission electron microscope. The physiological and biochemical characteristics such as the utilization of different carbon sources and enzymatic properties were determined by China Center for Type Culture Collection (CCTCC).

Cell growth and nicotine degradation
Culture temperature, pH, nicotine concentration and rotation rates were studied in order to identify the optimal conditions for cell growth and nicotine transformation. To determine the optimal temperature for cell growth, strain N1 was incubated in minimal medium containing 1 g l 21 nicotine at 23uC, 26uC, 30uC, 34uC, and 37uC with the initial pH set at 7.0. The optimal pH was determined by culturing N1 at pH values of 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, and 7.5. The pH was adjusted using 100 mmol phosphorous buffer. Once the optimal temperature and pH were determined, the effect of nicotine concentration (0.5, 1.0, 1.5, and 2.0 g l 21 ) was investigated under optimal temperature and pH. To evaluate the influence of the rotation rate of the reciprocal shaker on nicotine degradation, strain N1 was cultivated at different shaking rates of 0, 120, 180, and 220 rpm. The optimized conditions were then used for subsequent work.
During the incubation period, aliquots of the culture medium were sampled at the pre-determined intervals and analyzed at 600 nm by using a 2100 spectrophotometer (Unic Company, Shanghai). The samples were also preserved at 220uC for HPLC and UV absorption analyses.

Degradation of nicotine by strain N1
P. geniculata N1 was cultured under optimal conditions in ''nic medium'', Luria-Bertani (LB) medium and LB medium with 1 g l 21 nicotine, harvested during the late-exponential phase by centrifugation at 6,0006 g for 8 min at 4uC, and washed twice by sodium phosphate buffer (100 mM, pH 7.0). Then the cells were suspended in deionized water (OD 600 nm ,5) for reaction (called resting cells). Resting cells were resuspended in prepared PBS buffer (pH 7.0), and adjusted to OD 600 nm ,15, with the addition of 10% glycerol, 1 mM DTT and 2.5 mM PMSF. After sonification in the condition of 5 s on, 5 s off, 90 cycles, cell lysates were centrifuged at 12,000 rpm for 20 min, and the supernatant liquid were used for reaction (called crude cells). The nicotine degradation assay was performed at 30uC on a shaker rotating at 180 rpm.

Identification of metabolites in nicotine degradation
After the ''resting cell reaction'', the reaction mixture (1 ml) was evaporated until its dry at 50uC under the reduced pressure, and then dissolved in 200 ml of acetonitrile. The resulting solution was transferred to a vial and dried under nitrogen stream. Samples were analyzed using a GC-MS system (GCD 1800C, Hewlett-Packard) equipped with a flame ionization detector and a 50-mlong J&W DB-5MS column (Folsom, CA, USA) at 140uC. The injection port and detector were set at 260uC and 280uC, respectively. LC-MS analysis was performed by Agilent 1290 (ultra-performance liquid chromatography, UPLC) coupled with an Agilent 6230 electrospray ionization-time-of-flight-mass spectrometry (ESI-TOF-MS) with methanol-0.1% formic acid mixture

General analytical techniques
The nicotine present in the culture medium was quantified by HPLC (Agilent 1200 series) equipped with an Eclipse XDB-C18 column (column size, 25064.6 mm; particle size, 5 mm; Agilent). A mixture of methanol-1 mmol H 2 SO 4 (5:95 v v 21 ) was used as the mobile phase, at a flow rate of 0.5 ml min 21 . Qualitative analysis of nicotine and metabolites was carried out by UV-2500 spectrophotometer (Shimadzu).

Nucleotide sequence accession number
The nucleotide sequence reported in the present study has been deposited in the GenBank under the accession number JN607239.

Isolation and identification of strain N1
The isolated strain N1, which can utilize nicotine as the sole source of carbon and nitrogen, has been deposited at CCTCC under the accession number M2011183. Strain N1 forms small, circular, and convex colonies with neat edges on nicotine agar ( Figure S1A). Notably, the colonies were yellow, which is rarely observed in case of nicotine-degrading strains. The strain was identified as a non-spore-forming, gram-negative rod (0.561.5 mm) with 2 or 3 flagella at one pole. The image of strain N1 is presented in Figure S1B. Strain N1 could utilize a narrow range of carbon sources such as polychrom, and it grew weakly in glucose, amygdalin, arbutin, and saligenin ( Table 1). The physiological and biochemical characteristics, performed at CCTCC, are shown in Tables 1 and 2. Strain N1 incubated at 30uC showed the following characteristics: growth at 5% sodium chloride; positive for catalase and oxidase; positive for arginine dihydrogenase and lysine decarboxylase; negative for ornithine decarboxylase; utilization of citric acid or polychrome as the sole source of carbon for growth; positive for lipase (C14); negative for H 2 S and indole production; and negative for Voges-Proskauer test ( Table 2). The characteristics of strain N1 were strikingly similar to those of previously reported Pseudomonas geniculata strains [20].
The 16S rRNA sequence exhibits 99% identity with Pseudomonas and Stenotrophomonas species. Phylogenetic tree of 16S rRNA from 35 different strains is constructed using the molecular evolutionary genetics analysis tool (MEGA4.1) by neighbor joining (NJ) method and repeated bootstrapping for 1000 times was performed. Strain N1 is closest to the ortholog from Pseudomonas geniculata strain ATCC 19374T (Figure 1). In conclusion, strain N1 was classified as Pseudomonas geniculata based on the above results.

Cell growth and nicotine degradation
The effects of temperature on strain N1 is shown in Figure 2A and 2B. Temperature has a dramatic influence on the growth of N1, which showed the maximum rate of growth and nicotine degradation at 30uC. The growth rate was much slower with a gradual drop in temperature. Notably, little-to-no growth was observed at 34uC, which indicates a narrow tolerance range for temperature. Figure 2C and 2D show the impact of pH on the growth of strain N1. The data show that strain N1 could grow at the pH values ranging from 5.5 to 7.0. Thus, strain N1 prefers weak acidic environment, ranging from pH 6.0 to 6.5. The rate of cell growth dropped remarkably, when pH was ,6.0. However, the maximum biomass did not show a major difference. With an increase in the pH value, the cell growth rate dropped slightly in a neutral environment; no growth was detected in an alkaline environment. It should also be noted that the influence of pH on nicotine degradation was not significant. The degradation rate was rather stable at pH values ranging from 6.0 to 7.5, whereas it was much slower at a pH of 5.5, which was in line with the pattern of cell growth. Figure 2E and 2F illustrate the nicotine tolerance of strain N1. Strain N1 could grow well when the nicotine concentration was ,2 g l 21 . Moreover, with an increase in the concentration of nicotine in the growth medium, the maximum biomass increased proportionally. However, the growth was much slower when the initial nicotine concentration was 1.5 g l 21 rather than 1.0 g l 21 .
The maximum biomass was noted after 4.5 days, when the nicotine concentration was 1.0 g l 21 , while it took .8 days to reach the stationary phase in the presence of 1.5 g l 21 nicotine.
As shown in Figure 2G and 2H, the rotary rate of the shaker can impact cell growth by altering oxygen supply. The growth was extremely slow with low maximum biomass when the growth cultures were kept stationary. This finding confirmed our initial results, which identified strain N1 as an aerobic bacterium. The optimal rotary rate of 120 rpm min 21 resulted in the maximum growth rate and maximum biomass production. In conclusion, P. geniculata N1 grows best at 30uC, pH 6.5, and 120 rpm min 21 with a maximum nicotine-tolerating capability of 1.5 g l 21 (Figure 3).

Nicotine degradation by resting and crude cells
Resting cells harvested from nicotine medium (see materials and methods) were able to degrade 3 g l 21 nicotine within 3 h. As shown in Figure 4, the decrease in nicotine concentration and the formation of new peaks in the UV absorption spectrum ( Figure 4A) or HPLC spectrum ( Figure 4B and Figure S2) suggest the degradation of nicotine and generation of new metabolites. In contrast, the cells cultivated in LB medium did not exhibit the ability to degrade nicotine, illustrating that the enzymes required for nicotine degradation are inducible ( Figure S3). The crude cells of strain N1 harvested in nicotine medium were obtained after sonication in phosphate buffer (see materials and methods). However, the cell extracts could not degrade nicotine, and it is similar to the findings with strain Pseudomonas putida S16 [17] ( Figure S4). The phylogenetic tree is constructed using the molecular evolutionary genetics analysis tool (MEGA 4.1) by neighbor joining (NJ) method [19]. The repeated bootstrapping for 1,000 times was performed. doi:10.1371/journal.pone.0084399.g001

Nicotine biotransformation and metabolites identification
The culture broth of strain N1 was yellowish green, and blue color did not develop during nicotine biotransformation. This indicated that the nicotine-degradation pathway used differed from that used by Arthrobacter, Nocardioides and Rhodococcus strains. The GC-MS chromatogram is shown in Figure 5. The structures of compound A (nicotine, 12.603 min), B (myosmine, 13.581 min), and E (cotinine, 17.102 min) could be identified by comparing their mass spectra with the standard GC-MS spectral library ( Figure 5). Compound C (18.883 min) exhibited the following    Figure 6).

Discussion
The highly toxic alkaloid nicotine, present in tobacco waste, is removed from the environment via mineralization by bacteria.  Basic insights into the steps and intermediates of nicotine degradation by Arthrobacter and Pseudomonas species have been proposed and elucidated [4,18,21]. In this study, a novel nicotinedegrading bacterium N1 was isolated from tobacco leaves. The physiological and biochemical data show that the strain N1 belongs to the genus Pseudomonas. Most of the morphological and physiological traits of strain N1 were identical to those of Pseudomonas geniculata [20]. P. geniculata has been poorly reported in previously published literature, and this study is the first report demonstrating the nicotine-degrading ability of P. geniculata. In addition, it is interesting that strain N1 could utilize only a narrow range of carbon sources and efficiently degrade nicotine. Strain N1 may have a powerful membrane transport capacity; it has been reported to possess 28 multidrug efflux pump genes [22]. These fingdings imply a highly efficient nicotine uptake capacity of the strain and an efficient removal of end-products of nicotine catabolism from the cells which may help to explain the nicotine-degrading properties of strain N1.
Current understanding of nicotine degradation in bacteria is based on characterization of 6-hydroxynicotine (pyridine pathway) in Arthrobacter species [4] and N-methylmyosmine (pyrrolidine pathway) in Pseudomonas species [22][23][24][25]. Otherwise, Agrobacterium tumefaciens strain S33 could firstly transform nicotine to 6-hydroxy-N-methylmyosmine using pyridine pathway, and then further degrade 6-hydroxy-N-methylmyosmine to 6-hydrxoxy-3-succinoylpyridine and 2,5-dihydroxypyridine using pyrrolidine pathway [12]. In the present study, the formation of blue pigment was not observed during the transformation of nicotine by strain N1. Therefore, it can be proposed that the latter catabolic pathway of nicotine degradation is likely to be different from that of Arthrobacter. The intermediates 6-hydroxynicotine, 6-hydroxy-Nmethymyosime, 6-hydroxy-pseudooxynicotine, and 2,6-dihydroxypseudooxynicotine were identified by GC-MS and LC-MS analyses. The intermediates 6-hydroxy-3-succinoylpridine and 2,5-dihydroxy-pridine in P. putida S16 were not detected in the ''resting cell reactions'' of strain N1. It can be concluded that the upper pathway of nicotine degradation in strain N1 was similar to the pyridine pathway, and the further conversion of 2,6dihydroxypseudooxynicotine might be different from that ob-served in Arthrobacter and Pseudomonas. In addition, the intermediates myosime and cotinine can be detected by GC-MS. Thus, the direct demethylation to form myosime and the hydroxylation of nicotine at position 2 of pyrrolidine ring to form cotinine was proposed, and found to be similar to that of the strain Pseudomonas sp. CS3 [26].
In conclusion, it is proposed that the strain Pseudomonas geniculata N1 can decompose nicotine via a unique nicotine-degrading pathway.